Inhibitors of adp-ribosyl transferases, cyclases, and hydrolases

ABSTRACT

The present invention provides compounds having the formula: (I); Also provided are pro-drug compounds of the formula: (II); The invention also provides pharmaceutical compositions containing the above compounds, methods of using the above compounds as pharmaceuticals, and processes for preparing the above compounds. Methods for inhibiting an ADP-ribosyl transferase, ADP-ribosyl cyclase, ADP-ribosyl hydrolase, or NAD-dependent deacetylase enzyme using the above compounds, and methods for treating a disease or condition associated with an ADP-ribosyl transferase, ADP-‘ribosyl cyclase, ADP-ribosyl hydrolase, or NAD-dependent deacetylase enzyme in a subject using the above compounds are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/158,636, filed May 30, 2003.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of Grant Nos.GM19335 and AI34342 awarded by the National Institutes of Health.

BACKGROUND

(1) Field of the Invention

The present invention generally relates to inhibitors of ADP-ribosyltransferases, cyclases and hydrolases, and NAD-dependent deacetylases,including CD38. More specifically, the invention relates to improvedinhibitors of those enzymes, and inhibitor pro-drugs, where theinhibitors are designed according to the mechanism of the enzymes'action.

(2) Description of the Related Art

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CD38 is a membrane anchored homodimeric ectoenzyme common to a varietyof immune cells (Jackson and Bell, 1990) and other tissues (Fernandez etal., 1998) including pancreas (Kato et al., 1995) kidney (Khoo andChang, 2000) and brain (Mizuguchi et al., 1995). CD38 is homologous toBST-1 (Kaisho et al., 1994; Itoh et al., 1994), bone stromal cellantigen, and invertebrate ADP-ribosyl cyclases (Lee and Aarhus, 1991;States et al., 1992) and catalyzes the formation of cyclic-ADP-ribose(cADPR, Lee et al., 1994) from NAD⁺ (Scheme 1, Rusinke and Lee, 1989).cADPR is a potent second messenger that directly activates Ca⁺² releaseinside of cells via an IP₃ independent mechanism (Lee, 2001; Lee, 1995;Clapper et al., 1987) thought to be mediated by ryanodine receptors(Lee, 2001). Recent evidence indicates that cADPR and CD38 plays acrucial role in the human immune response by activation of thecell-mediated neutrophil response to bacterial infection(Partida-Sanchez et al., 2001) and associated inflammatory physiology(Id.; Normark et al., 2001). ADP-ribosyl-cyclase and cADPR signaling hasalso been demonstrated in plants as mediator of the abscisic acidactivated stress response (Wu et al., 1997).

Not surprisingly, the ADP-ribosyl cyclases have been targets forinhibitor design (Sleath et al., 1991; Muller-Steffner et al., 1992;Bethelier et al., 1998; Wall et al., 1998; Sauve et al., 2000). Also,analogs of cADPR with antagonistic (Sato et al., 1999a; Sato et al.,1999b; Hara-Yokoyama et al., 2001; Walseth and Lee, 1993), or agonistic(Sethi et al., 1997; Walseth et al., 1993; Ashamu et al., 1997; Galioneet al., 1997; Wong et al., 1999; Lee and Aarhus, 1998; Baily et al.,1996) properties have been reported. Most of the inhibitors and cADPRanalogs are phosphorylated compounds (Lee, 2001), and have practicallimitations affecting their use in whole cell or whole tissueinvestigations, because of the difficulty of passing charges across cellmembranes (Id.). Although altered inhibitor structure to nucleosidescould potentially make compounds more cell permeant, no reports ofnucleoside-based CD38 or ADP-ribosyl-cyclase inhibitors have appeared.

In prior work, the mononucleotide ara-F-NMN⁺ was shown to be a potentinhibitor of CD38 with a K_(i) value of 61 nM (Sauve et al., 2000). ThisK_(i) is similar to the dinucleotide inhibitor ara-F-NAD⁺ (Sleath etal., 1991), where a K_(i) value of 169 nM was reported (Muller-Steffneret al., 1992).

In other work, mechanism-based inhibitors of ADP-ribosyl transferases,cyclases and hydrolases, and NAD-dependent deacetylases were found tohave several advantages to the above nucleotide-based inhibitors (U.S.patent application Ser. No. 10/038,760, incorporated by reference in itsentirety). Those inhibitors react rapidly to form a covalentintermediate that cannot cyclize and that are relatively stable tohydrolysis, thereby trapping the enzyme in a catalytically-inactiveform. Further development of these mechanism-based inhibitors to providehighly stable, potent inhibitors of ADP-ribosyl transferases, cyclasesand hydrolases, and NAD-dependent deacetylases is desirable.

SUMMARY OF THE INVENTION

Accordingly, the inventors have discovered that providing anelectron-contributing moiety to the leaving group of the mechanism-basedinhibitors described in U.S. patent application Ser. No. 10/038,760 (the'760 application) greatly stabilizes the compounds to hydrolysis. Theresulting improved inhibitors provide greater potential for therapeuticbenefits, and provides improved reagents for studying ADP-ribosyltransferases, cyclases and hydrolases, and NAD-dependent deacetylases,including CD38. Pro-drug compounds of the inhibitors have also beendeveloped.

Thus, in some embodiments, the present invention is directed tocompounds represented by the formula:

where A is a nitrogen-, oxygen-, or sulfur-linked aryl, alkyl, cyclic,or heterocyclic group. In these embodiments, the group A is furthersubstituted with an electron contributing moiety. Additionally, B ishydrogen, or a halogen, amino, or thiol group; C is hydrogen, or ahalogen, amino, or thiol group; and D is a primary alcohol, a hydrogen,or an oxygen, nitrogen, carbon, or sulfur linked to phosphate, aphosphoryl group, a pyrophosphoryl group, or adenosine monophosphatethrough a phosphodiester or carbon-, nitrogen-, or sulfur-substitutedphosphodiester bridge, or to adenosine diphosphate through aphosphodiester or carbon-, nitrogen-, or sulfur-substitutedpyrophosphodiester bridge. The compounds are preferably inhibitors ofADP-ribosyl transferase, ADP-ribosyl cyclase, ADP-ribosyl hydrolase,and/or NAD-dependent deacetylase enzymes.

The invention is also directed to pro-drug compounds represented by theformula:

where A is a nitrogen-, oxygen-, or sulfur-linked aryl, alkyl, cyclic,or heterocyclic group; B is hydrogen, or a halogen, amino, or thiolgroup; C is hydrogen, or a halogen, amino, or thiol group; D is theester —OOCR where R is an alkyl or an aryl, a primary alcohol, ahydrogen, or an oxygen, nitrogen, carbon, or sulfur linked to phosphate,a phosphoryl group, a pyrophosphoryl group, or adenosine monophosphatethrough a phosphodiester or carbon-, nitrogen-, or sulfur-substitutedphosphodiester bridge, or to adenosine diphosphate through aphosphodiester or carbon-, nitrogen-, or sulfur-substitutedpyrophosphodiester bridge; and E is OH or the ester —OOCR where R is analkyl or an aryl. In these embodiments, at least one of D or E is theester —OOCR where R is an alkyl or an aryl.

Pharmaceutical compositions comprising the above compounds in apharmaceutically acceptable carrier are also encompassed by theinvention.

In other embodiments, the invention is directed to methods forinhibiting an ADP-ribosyl transferase, ADP-ribosyl cyclase, ADP-ribosylhydrolase or an NAD-dependent deacetylase enzyme. The methods comprisecontacting the enzyme with an amount of any of the above compoundseffective to inhibit the enzyme.

Additionally, the invention is directed to methods for treating adisease or condition associated with an ADP-ribosyl transferase,ADP-ribosyl cyclase, ADP-ribosyl hydrolase, or NAD-dependent deacetylaseenzyme in a subject in need of treatment thereof. These methods compriseadministering to the subject any of the above-described inhibitor orpro-drug compounds in an amount effective to treat the disease orcondition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the structure of inhibitors 1-3.

FIG. 2 provides graphs illustrating data measuring time courses ofinhibition of CD38 by different concentrations of 1 as assayed byconversion of NGD⁺ to cGDPR (100 μM NGD⁺). Panel A shows the initialrates of reaction of curves from Panel B were fit to the equation forcompetitive inhibition to determine K_(i) in Table 2. Panel B shows theextended time courses of two-phase inhibition of CD38 by differentconcentrations of 1 as assayed by conversion of NGD⁺ to cGDPR. Inhibitorconcentrations are shown. The solid lines represent the best fit to theslow-onset equation given in the text. The rate constant (k₁) for theslow phase derived from these curves is 4.2×10⁻³ s⁻¹.

FIG. 3 provides a graph illustrating data measuring time course oftwo-phase inhibition of CD38 by different concentrations of 1 as assayedby conversion of NGD⁺ to cGDPR (40 μM NGD⁺). Inhibitor concentrationsare shown on the right of curves. The solid lines represent the best fitto the slow-onset equation given in the text. The rate constant (k_(on))for the slow phase derived from these curves is 8.3×10⁻³ s⁻¹. Theinitial rates from these curves were used to determine K_(i) (Table 2).

FIG. 4 provides a graph of data from a recovery experiment to measurerate of recovery of CD38 from inhibition by 1 in the presence of excessNGD⁺. The top curve is a control, of uninhibited enzyme. The bottomcurve shows the recovery process as increasing free CD38 generatesincreasing rates of cGDPR formation. The solid curve represents the bestfit to the recovery equation described in the text. The recovery ratedetermined was 2×10⁻⁵ s⁻¹.

FIG. 5 provides a graph illustrating data measuring rates of CD38recovery as a function of nicotinamide concentration as determined bystopped flow. The apparent Michaelis parameters were derived from thebest fit of the points to the Michaelis-Menten equation. The parameterk_(base) is defined as in Scheme 7.

FIG. 6 provides HPLC chromatograms of base exchange reaction solutions.Panel A shows an initial chromatogram at 0 time containing 1 μM CD38, 75μM 2, and 20 mM nicotinamide. Panel B shows a chromatogram of the samesolution after several hours of incubation at 19° C. showing theappearance of the base exchanged product β-nicotinamide-deoxyriboside(Jackson and Bell, 1990). Abbreviations: β5MeNdR;β-5-methylnicotinamide-deoxyriboside, βNdR;β-nicotinamide-deoxyriboside.

FIG. 7 provides a graph illustrating data showing a steady state rate ofbase exchange of CD38 in which 2 forms 1 by reaction with nicotinamide.The Michaelis parameters were derived from the best fit of the points tothe Michaelis-Menten equation.

FIG. 8 provides a graph showing radiochemical labeling of one nanomoleof CD38 (monomer) by [2-³H]-1 as measured by gel filtration andscintillation counting. The solid curve represents the best fit to theequation P=A+A₂ exp(−kt). The curve obtains a value of k of 0.01 s⁻¹.Specific radioactivity of inhibitor is 866 cpm/nmol.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides improvements to the mechanism-basedinhibitors of ADP-ribosyl cyclases, ADP-ribosyl hydrolases, ADP-ribosyltransferases, and NAD-dependent deacetylases first disclosed in U.S.patent application Ser. No. 10/038,760 (the '760 application). Theimprovements are based in part on the discovery that the stability ofthe inhibitors can be improved by substituting the leaving group of theinhibitors with an electron-contributing moiety. Without being limitedto any particular mechanism for the improved stability, it is believedthat the electron-contributing moiety improves stability of theinhibitors by causing a decrease in the hydrolysis of the leaving groupfrom the rest of the inhibitor.

Thus, in one aspect, the present invention provides compounds having theformula:

In these embodiments, A is a nitrogen-, oxygen-, or sulfur-linked aryl,alkyl, cyclic, or heterocyclic group. The A moieties thus described haveleaving group characteristics. In embodiments encompassed by the presentinvention, A is further substituted with an electron contributingmoiety. Additionally, B and C are hydrogen, or either B or C is ahalogen, amino, or thiol group and the other of B or C is hydrogen; andD is a primary alcohol, a hydrogen, or an oxygen, nitrogen, carbon, orsulfur linked to phosphate, a phosphoryl group, a pyrophosphoryl group,or adenosine monophosphate through a phosphodiester or carbon-,nitrogen-, or sulfur-substituted phosphodiester bridge, or to adenosinediphosphate through a phosphodiester or carbon-, nitrogen-, orsulfur-substituted pyrophosphodiester bridge.

Preferably, A is a substituted N-linked aryl or heterocyclic group, anO-linked aryl or heterocyclic group having the formula —O—Y, or anS-linked aryl or heterocyclic group having the formula —O—Y, both B andC are hydrogen, or either B or C is a halogen, amino, or thiol group andthe other of B or C is hydrogen; and D is a primary alcohol or hydrogen.Nonlimiting preferred examples of A are set forth in Table 1, where eachR is H or an electron-contributing moiety and Z is an alkyl, aryl,hydroxyl, OZ′ where Z′ is an alkyl or aryl, amino, NHZ′ where Z′ is analkyl or aryl, or NHZ′Z″ where Z′ and Z″ are independently an alkyl oraryl. TABLE 1

More preferably, A is a substituted nicotinamide group (Table 1, i,where Z is H), a substituted pyrazolo group (Table 1, vii), or asubstituted 3-carboxamid-imidazolo group (Table 1, viii, where Z is M.Additionally, preferably, both B and C are hydrogen, or either B or C isa halogen, amino, or thiol group and the other of B or C is hydrogen;and D is a primary alcohol or hydrogen.

Without being bound to any particular mechanism, it is believed that theelectron-contributing moiety on A stabilizes the compounds of theinvention such that they are less susceptible to hydrolysis from therest of the compound. For example, the compoundβ-1′-5-methyl-nicotinamide-2′-deoxyribose (established as an effectiveinhibitor of CD38 in Example 1) was compared withβ-1′-nicotinamide-2′-deoxyribose in its ability to resist solutionhydrolysis. The measured rate constant for solution hydrolysis (10 mMpotassium phosphate, pH 6.5, 25° C.) of β-1′-nicotinamide-2′-deoxyribosewas 9.6×10⁻⁵ s⁻¹ whereas the rate of solution hydrolysis ofβ-1′-5-methyl-nicotinamide-2′-deoxyribose was measured at 1.5×10⁻⁵ s⁻¹,demonstrating that the methyl group on the nicotinamide-2′-deoxyribosidecaused a decrease in the rate of hydrolysis by a factor of 6. Thisdifference in chemical stability means thatβ-1′-nicotinamide-2′-deoxyribose is 50% depleted from solution in 2hours, whereas β-1′-5-methyl-nicotinamide-2′-deoxyribose is nothydrolyzed by 50% until 12 hours. This improved chemical stabilityimproves the value of the compound, since it is available for action forlonger periods of time in biological systems due to resistance tohydrolytic breakdown.

The skilled artisan could envision many electron-contributing moietiesthat would be expected to serve this stabilizing function. Nonlimitingexamples of suitable electron contributing moieties are methyl, ethyl,O-methyl, amino, NMe₂, hydroxyl, CMe₃, aryl and alkyl groups.Preferably, the electron-contributing moiety is a methyl, ethyl,O-methyl, amino group. In the most preferred embodiments, theelectron-contributing moiety is a methyl group.

It is also preferred that, in addition to the electron-contributingmoiety, the A group also comprises a carboxamid (CONH₂) group, as innicotinamide, as it is believed that the carboxamid group improves theability of the compound to be inhibitory to ADP-ribosyl cyclases,ADP-ribosyl hydrolases, ADP-ribosyl transferases, and/or NAD-dependentdeacetylases, such as CD38.

In some embodiments, A has two or more electron contributing moieties.

Some preferred examples of the compounds of the invention are providedas compounds I, II, and III below.

wherein Z is an alkyl, aryl, hydroxyl, OZ′ where Z′ is an alkyl or aryl,amino, NHZ′ where Z′ is an alkyl or aryl, or NHZ′Z″ where Z′ and Z″ areindependently an alkyl or aryl; E and F are independently H, CH₃, OCH₃,CH₂CH₃, NH₂, OH, NHCOH, NHCOCH₃, N(CH₃)₂, C(CH₃)₂, an aryl or a C3-C10alkyl, preferably provided that, when either of E or F is H, the otherof E or F is not H;

wherein G, J or K is CONHZ, Z is an alkyl, aryl, hydroxyl, OZ′ where Z′is an alkyl or aryl, amino, NHZ′ where Z′ is an alkyl or aryl, or NHZ′Z″where Z′ and Z″ are independently an alkyl or aryl, and the other two ofG, J and K is independently CH₃, OCH₃, CH₂CH₃, NH₂, OH, NHCOH, NHCOCH₃;

wherein Z is an alkyl, aryl, hydroxyl, OZ′ where Z′ is an alkyl or aryl,amino, NHZ′ where Z′ is an alkyl or aryl, or NHZ′Z″ where Z′ and Z″ areindependently an alkyl or aryl; and L is CH₃, OCH₃, CH₂CH₃, NH₂, OH,NHCOH, NHCOCH₃.

In more preferred embodiments, the compound is formula I above, whereinE and F are independently H, CH₃, OCH₃, or OH, preferably provided that,when either of E or F is H, the other of E or F is not H.

In even more preferred embodiments, the compound isβ-1-5-methyl-nicotinamide-2′-deoxyribose,β-D-1-5-methyl-nicotinamide-2′-deoxyribofuranoside,β-1′-4-methyl-nicotinamide-2′-deoxyribose,β-D-1′-4-methyl-nicotinamide-2′-deoxyribofuranoside,β-1′-4,5-dimethyl-nicotinamide-2-deoxyribose orβ-D-1′-4,5-dimethyl-nicotinamide-2′-deoxyribofuranoside.

In the most preferred embodiment, the compound isβ-1′-5-methyl-nicotinamide-2′-deoxyribose.

Preferably, the compounds of the present invention are inhibitors ofADP-ribosyl cyclases, ADP-ribosyl hydrolases, ADP-ribosyl transferases,and/or NAD-dependent deacetylases, such as CD38. See Example 1.

Even though it is preferred that the compounds are inhibitors ofADP-ribosyl cyclases, ADP-ribosyl hydrolases, ADP-ribosyl transferases,and/or NAD-dependent deacetylases, the forms of the compounds that arenot inhibitors are also useful, for example as a negative control instudies of the effectiveness of the inhibitor for therapeutic purposes.

Methods for determining the inhibitory activity of any particularcompound are routine. Inhibitory activity of the compounds disclosedherein can be determined by standard assays known in the art. Forexample, the enzyme may be incubated with the inhibitor and a substrateof the enzyme, and absorbance then may be monitored, as described below.Additionally, the enzyme may be incubated with a radioactive inhibitor,and radiochemical measurements of reaction rates may be taken, asdescribed below. Slow-onset inhibitor binding may be determined usingmethods such as those described in Merkler et al., 1990.

Molecules of the novel class of mechanism-based inhibitors disclosedherein accomplish mechanism-based trapping at the catalytic site oftheir target enzymes. The inhibitor is designed to react rapidly to forma covalent intermediate that cannot cyclize and that is relativelystable to hydrolysis, thereby trapping the enzyme in acatalytically-inactive form. For example, as elaborated in Example 1,the novel inhibitor β-D-1′-5-methyl-nicotinamide-2′-deoxyribose acts asa reversible competitive inhibitor (K_(i)=4.0 μM) of CD38, and isfollowed by slow-onset inactivation of the enzyme. Inactivated enzyme iscovalently modified by the deoxyriboside. Active CD38 is slowlyregenerated by hydrolysis in the absence of added substrates, and israpidly regenerated in the presence of excess nicotinamide. Theseproperties of inhibitor action give rise to an effective inhibitionconstant of 12.5 nM. This novel class of mechanism based inhibitors haspotential for the regulation of cyclic ADP-ribose levels through CD38,and provides new tools for investigating the various pathways in whichADP-ribosyl transferases, cyclases, and hydrolases, and NAD-dependentdeacetylases have been implicated.

The compounds of the present invention are useful both in free form andin the form of salts. The term “pharmaceutically acceptable salts” isintended to apply to non-toxic salts derived from inorganic or organicacids and includes, for example, salts derived from the following acids:hydrochloric, sulfuric, phosphoric, acetic, lactic, fumaric, succinic,tartaric, gluconic, citric, methanesulfonic, and p-toluenesulfonicacids.

Also provided are compounds that are the tautomers,pharmaceutically-acceptable salts, esters, and pro-drugs of theinhibitor compounds disclosed herein.

The biological availability of the compounds of the invention can beenhanced by conversion into a pro-drug form. Such a pro-rug can haveimproved lipophilicity relative to the unconverted compound, and thiscan result in enhanced membrane permeability. One particularly usefulform of pro-drug is an ester derivative. Its utility relies upon theaction of one or more of the ubiquitous intracellular lipases tocatalyse the hydrolysis of ester groups, to release the active compoundat or near its site of action. In one form of pro-drug, one or morehydroxy groups in the compound (for example, the 3′ hydroxy of adeoxyribose group, or a hydroxy group at position D) can be O-acylated,to make an acylate derivative. In preferred embodiments, the pro-drughas the formula IV,

where X and Y are independently an alkyl, heteroatom or heterogroup, orH; and R is an alkyl or aryl (see Example 2).

Thus, in some embodiments, the invention is directed to a pro-drugrepresented by the formula:

where A is a nitrogen-, oxygen-, or sulfur-linked aryl, alkyl, cyclic,or heterocyclic group; B is hydrogen, or a halogen, amino, or thiolgroup; C is hydrogen, or a halogen, amino, or thiol group; D is theester —OOCR where R is an alkyl or an aryl, a primary alcohol, ahydrogen, or an oxygen, nitrogen, carbon, or sulfur linked to phosphate,a phosphoryl group, a pyrophosphoryl group, or adenosine monophosphatethrough a phosphodiester or carbon-, nitrogen-, or sulfur-substitutedphosphodiester bridge, or to adenosine diphosphate through aphosphodiester or carbon-, nitrogen-, or sulfur-substitutedpyrophosphodiester bridge; and E is OH or the ester —OOCR where R is analkyl or an aryl. In these embodiments, at least one of D or E is theester —OOCR where R is an alkyl or an aryl.

Preferably, the pro-drug compounds of these embodiments inhibits atleast one enzyme selected from the group consisting of an ADP-ribosyltransferase, an ADP-ribosyl cyclase, an ADP-ribosyl hydrolase, and anNAD-dependent deacetylase enzyme, when the compound is treated with anesterase. In the most preferred embodiments, the enzyme is a CD38.

In other preferred embodiments, A is further substituted with anelectron contributing moiety, preferably a methyl, ethyl, O-methyl,amino, NMe₂, hydroxyl, CMe₃, aryl or C3-C10 alkyl; more preferably amethyl, ethyl, O-methyl or amino. In the most preferred embodiments, theelectron contributing moiety is a methyl. A may also comprise a secondelectron contributing moiety.

As with the analogous inhibitor compounds described above, A ispreferably capable of base exchange with nicotinamide in the presence ofa CD38.

In the most preferred embodiments, both D and E are the ester —OOCRwhere R is an alkyl or an aryl; in other preferred embodiments, A is anN-linked aryl or heterocyclic group, preferably a substitutednicotinamide, pyrazolo, or imidazolo group. In more preferredembodiments, the pro-drug compound is amethyl-nicotinamide-2′-deoxyriboside ester, preferably a5-methyl-nicotinamide-2′-deoxyriboside ester or a4-methyl-nicotinamide-2′-deoxyriboside ester, more preferably an esterof β-1′-5-methyl-nicotinamide-2′-deoxyribose,β-D-1′-5-methyl-nicotinamide-2′-deoxyribofuranoside,β-1′-4-methyl-nicotinamide-2′-deoxyribose,β-D-1′-4-methyl-nicotinamide-2′-deoxyribofuranoside,β-1′-4,5-dimethyl-nicotinamide-2′-deoxyribose orβ-D-1′-4,5-dimethyl-nicotinamide-2′-deoxyribofuranoside. In the mostpreferred embodiments the pro-drug compound is an ester ofβ-1′-5-methyl-nicotinamide-2′-deoxyribose orβ-1′-4-methyl-nicotinamide-2′-deoxyribose.

In other embodiments of these pro-drug compounds, A is an O-linked arylor heterocyclic group having the formula —O—Y, where Y is an aryl orheterocyclic group; in additional embodiments, A is an S-inked aryl orheterocyclic group having the formula —S—Y, where Y is an aryl orheterocyclic group. In further embodiments, both B and C are hydrogen,or either B or C is a halogen, amino, or thiol group and the other of Bor C is hydrogen. In still further embodiments, D is a primary alcoholor hydrogen.

Pro-drug forms of a 5-phosphate ester derivative of compounds of thecompounds of the present invention can also be made. These may beparticularly useful, since the anionic nature of the 5-phosphate maylimit its ability to cross cellular membranes. Conveniently, such a5-phosphate derivative can be converted to an unchargedbis(acyloxymethyl) ester derivative. The utility of such a pro-drugrelies upon the action of one or more of the ubiquitous intracellularlipases to catalyse the hydrolysis of ester groups, releasing a moleculeof formaldehyde and a compound of the present invention at or near itssite of action. Specific examples of the utility of, and general methodsfor making, such acyloxymethyl ester pro-drug forms of phosphorylatedcarbohydrate derivatives have been described (Kang et al., 1998; Jianget al., 1998; Li et al., 1997; Kruppa et al., 1997).

In another aspect, the present invention provides pharmaceuticalcompositions comprising a pharmaceutically effective amount of any ofthe inhibitor or pro-drug compounds described above. Preferably, thepharmaceutical compositions comprise an inhibitor or pro-drug compoundchosen from the preferred compounds described above.

In the pharmaceutical compositions of the present invention, thepharmaceutically-acceptable carrier must be “acceptable” in the sense ofbeing compatible with the other ingredients of the composition, and notdeleterious to the recipient thereof. Examples of acceptablepharmaceutical carriers include carboxymethyl cellulose, crystallinecellulose, glycerin, gum arabic, lactose, magnesium stearate, methylcellulose, powders, saline, sodium alginate, sucrose, starch, talc, andwater, among others. Formulations of the pharmaceutical composition maybe conveniently presented in unit dosage.

The formulations of the present invention may be prepared by methodswell-known in the pharmaceutical art. For example, the inhibitor orpro-drug compound may be brought into association with a carrier ordiluent, as a suspension or solution. Optionally, one or more accessoryingredients (e.g., buffers, flavoring agents, surface active agents, andthe like) also may be added. The choice of carrier will depend upon theroute of administration.

The pharmaceutical compositions are useful for administering theinhibitor or pro-drug composition of the present invention to a subjectto treat a disease or condition associated with an ADP-ribosyltransferase, ADP-ribosyl cyclase, ADP-ribosyl hydrolase, orNAD-dependent deacetylase enzyme, including any of those describedabove. The inhibitor or pro-drug compound is provided in an amount thatis effective to treat a disease or condition associated with anADP-ribosyl transferase, ADP-ribosyl cyclase, ADP-ribosyl hydrolase, orNAD-dependent deacetylase enzyme in the subject. That amount may bereadily determined by the skilled artisan, as described above.

According to another aspect of the present invention, there is provideda method for inhibiting an ADP-ribosyl transferase, ADP-ribosyl cyclase,ADP-ribosyl hydrolase, or NAD-dependent deacetylase enzyme. As usedherein, “ADP-ribosyl transferase” refers to those enzymes which catalyzethe transfer of ADP-ribose (adenosme 5″-diphospho-5′-a-D-ribose) fromNAD⁺ (nicotinamide adenine dinucleotide) to acceptor groups that arechemically reactive as nucleophiles, as well as enzymes that sharecatalytic site homology with such enzymes. The acceptor groups includethe nucleophilic groups of proteins, nucleic acids, sugars, and lipids.Biologically reactive nucleophiles also include other metabolitescontaining carboxyl groups, amino groups, guanidinium groups, thiolgroups, and nitrogens of aromatic or aliphatic compounds, as well asother groups chemically recognized as having nucleophilic character. TheADP-ribosyl transferase family of enzymes produces ADP-ribosylatedproteins, ADP-ribosylated nucleic acids, ADP-ribosylated sugars, sugarpolymers in homo- or hetero-polymeric forms, glycoproteins,ADP-ribosylated lipids, and ADP-ribosylated compounds of cellularmetabolism. Compounds of cellular metabolism include carboxylic acids,sugars, amino acids, lipids, nucleotides, nucleosides, vitamins, andintermediates in the biochemical pathways that synthesize thesecompounds of cellular metabolism.

As used herein, “ADP-ribosyl cyclase” includes those enzymes thatcatalyze the conversion of NAD⁺ to ADP-ribose (adenosine5″-diphospho-5′-a-D-ribose), in which reaction a chemical bond betweencarbon 1′ of the a-D-ribose group of NAD⁺ (nicotinamide adeninedinucleotide) is transferred to any nucleophilic acceptor group withinthe same ADP-ribose molecule, thereby forming a cyclic ring system notexisting in the parent molecule of NAD⁺. Also included are enzymes thatshare catalytic site homology with such ADP-ribosyl cyclase enzymes.Nucleophilic acceptor groups include nitrogen and oxygen groups of theparent NAD⁺ molecule (e.g., the structure of cyclic-ADP-ribose, in whichthe carbon 1′ of the a-D-ribose group of NAD⁺ is cyclized to nitrogen 1′of the adenine ring to form a new cyclic ring).

Additionally, as used herein, “ADP-ribosyl hydrolase” refers to thoseenzymes that catalyze the transfer of ADP-ribose (adenosine5″-diphospho-5′-α-D-ribose) from NAD⁺ (nicotinamide adeninedinucleotide) in the formation of ADP-ribose or cyclic-ADP-ribose.“ADP-ribosyl hydrolase”, as used herein, also includes enzymes thatcatalyze the removal of ADP-ribose, in a hydrolytic reaction, from theADP-ribosylated groups that are chemically reactive as nucleophiles,defined above. Also included are enzymes that share catalytic sitehomology with such ADP-ribosyl hydrolase enzymes. ADP-ribosylated groupsthat are chemically reactive as nucleophiles include the groups ofADP-ribosylated-proteins, ADP-ribosylated-nucleic acids,ADP-ribosylated-sugars, and ADP-ribosylated-lipids from the covalentADP-ribose. Biologically reactive groups removed from ADP-ribose byhydrolysis may also include biological metabolites containingADP-ribosylated-carboxyl groups, ADP-ribosylated-amino groups,ADP-ribosylated-guanidinium groups, ADP-ribosylated-thiol groups,ADP-ribosylated-nitrogens of aromatic or aliphatic compounds, and otherADP-ribosylated groups chemically recognized as having nucleophiliccharacter. This family of hydrolases regenerates proteins fromADP-ribosylated proteins, nucleic acids from ADP-ribosylated nucleicacids, sugars from ADP-ribosylated sugars, sugar polymers in homo- orhetero-polymeric forms from their ADP-ribosylated states, andglycoproteins from ADP-ribosylated glycoproteins, lipids fromADP-ribosylated lipids, and removes ADP-ribose from ADP-ribosylatedcompounds of cellular metabolism. Compounds of cellular metabolisminclude carboxylic acids, sugars, amino acids, lipids, nucleotides,nucleosides, vitamins, and intermediates in the biochemical pathwaysthat synthesize these biological metabolites.

Examples of ADP-ribosyl transferases, cyclases, and hydrolases, andNAD-dependent deacetylases include, without limitation, NAD-dependentdeacetylases involved in the regulation of gene expression (e.g., SIRfamily enzymes and their homologues, human CD38, the human ADP-ribosylcyclase, invertebrate and plant ADP-ribosyl cyclases [e.g., Aplysiacalifornica ADP ribosyl-cyclase], and human bone stromal cell antigen[humBST1]). Preferably, the enzyme of the present invention is CD38.

The method for inhibiting an ADP-ribosyl transferase, ADP-ribosylcyclase, ADP-ribosyl hydrolase, or NAD-dependent deacetylase comprisescontacting an ADP-ribosyl transferase, ADP-ribosyl cyclase, ADP-ribosylhydrolase, or NAD-dependent deacetylase enzyme with one of thepreviously described inhibitor compounds or theirpharmaceutically-acceptable salts in an amount effective to inhibit theenzyme. In other embodiments, the enzyme is inhibited by any of thepreviously described pro-drug compositions after treating the pro-drugcomposition with an esterase. The ADP-ribosyl transferase, ADP-ribosylcyclase, ADP-ribosyl hydrolase, or NAD-dependent deacetylase enzyme mayinclude any of those described above (e.g., ADP-ribosyl-transferasesinvolved in the regulation of gene expression [e.g., SIR family enzymesand their homologues], human CD38, the human ADP-ribosyl cyclase,invertebrate and plant ADP-ribosyl cyclases [e.g., Aplysia californicaADP ribosyl-cyclase], and human bone stromal cell antigen [humBST1]). Ina preferred embodiment, the enzyme is CD38. Preferably, the inhibitor isone of the preferred inhibitors previously described, in apharmaceutical composition.

As used herein, an “amount effective to inhibit the enzyme” refers to anamount that disables, disrupts, or inactivates the function of theenzyme. Inhibitor compounds contemplated for the inhibition ofADP-ribosyl transferase, ADP-ribosyl cyclase, ADP-ribosyl hydrolase, orNAD-dependent deacetylase enzymes may form a combination of enzyme andinhibitor, thereby generating complexes that reduce the catalyticfunction of the enzyme.

The inhibitor or pro-drug compound of the present invention, or apharmaceutically-acceptable salt thereof, may be contacted with theenzyme either in vivo or in vitro, using techniques well known to one ofskill in the art. Where contacting is effected in vitro, the inhibitoror pro-drug compound may be used as tools for investigating the pathwaysin which ADP-ribosyl transferase, ADP-ribosyl cyclase, ADP-ribosylhydrolase, or NAD-dependent deacetylase enzymes are involved. Wherecontacting is effected in vivo, the inhibitor or pro-drug compound maybe used to treat a disease or condition in which it is desirable todecrease the activity of an ADP-ribosyl transferase, ADP-ribosylcyclase, ADP-ribosyl hydrolase, or NAD-dependent deacetylase enzyme.

Accordingly, the present invention further provides methods for treatinga disease or condition that is directly or indirectly associated with anADP-ribosyl transferase, ADP-ribosyl cyclase, ADP-ribosyl hydrolase, orNAD-dependent deacetylase enzyme in a subject in need of treatmentthereof. These methods comprise administering to the subject any one ofthe previously described inhibitor or pro-dug compounds, or apharmaceutically-acceptable salt thereof, in an amount effective totreat the disease or condition. As used herein, a “subject” is a mammal,including, without limitation, a cow, dog, human, monkey, mouse, pig, orrat, as described above. Preferably, the subject is a human. TheADP-ribosyl transferase, ADP-ribosyl cyclase, ADP-ribosyl hydrolase, orNAD-dependent deacetylase enzyme may include any of those describedabove (e.g., NAD-dependent deacetylases involved in the regulation ofgene expression [e.g., SIR family enzymes and their homologues], humanCD38, the human ADP-ribosyl cyclase, invertebrate and plant ADP-ribosylcyclases [e.g. Aplysia californica ADP ribosyl-cyclase], and human bonestromal cell antigen [humBST1]). In one embodiment of the presentinvention, the enzyme is CD38.

As used herein, “disease” refers to any deviation from, or interruptionof, the normal structure or function of any part, organ, or system (orcombination thereof) of the body that presents an abnormal or pathologicbody state. As further used herein, “condition” refers to any state ofphysical or mental abnormality. Furthermore, as used herein, “a diseaseor condition associated with an ADP-ribosyl transferase, ADP-ribosylcyclase, ADP-ribosyl hydrolase, or NAD-dependent deacetylase enzyme”includes a disease or condition wherein an ADP-ribosyl transferase,ADP-ribosyl cyclase, ADP-ribosyl hydrolase, or NAD-dependent deacetylaseenzyme contributes to (either directly or indirectly), or is responsiblefor, the pathophysiology of the disease or condition, or in which it isdesirable to decrease the activity of an ADP-ribosyl transferase,ADP-ribosyl cyclase, ADP-ribosyl hydrolase, or NAD-dependent deacetylaseenzyme, or in which it is desirable to regulate the level of cADPR.

Inhibition of cADPR-stimulated calcium release is expected to havesignificant effects on calcium-mediated signaling pathways in many cellsand tissues. Accordingly, in the method of the present invention, thedisease or condition associated with an ADP-ribosyl transferase,ADP-ribosyl cyclase, ADP-ribosyl hydrolase, or NAD-dependent deacetylaseenzyme may include any disease or condition associated with a defect ordeficiency in the transmembrane flux of calcium (Ca²⁺) ions into or outof cells, particularly vascular smooth muscle cells, cardiac musclecells, and cells of the nervous system. Examples of such diseases mayinclude, without limitation, angina (e.g., angina pectoris, chronicstable angina, and vasospastic angina), arrhythmias, atrialfibrillation, hypertension, paroxysmal supraventricular tachycardia,acute disseminated encephalomyelitis (ADEM), acute transverse myelitis,acute viral encephalitis, adrenoleukodystrophy (ALD),adrenomyeloneuropathy, AIDS-vacuolar myelopathy, experimental autoimmuneencephalomyelitis (EAE), experimental autoimmune neuritis (EAN),HTLV-associated myelopathy, Leber's hereditary optic atrophy, multiplesclerosis (MS), progressive multifocal leukoencephalopathy (PML),subacute sclerosing panencephalitis, and tropical spastic paraparesis.

In mammals, CD38 and cADPR have been implicated in the regulation ofcellular processes, including insulin release (Okamoto et al., 1999),lymphocyte activation (Mehta et al., 1996; Cockayne et al., 1998), bonehomeostasis (Sun et al., 1999), neutrophil activation in response toacute bacterial (or pathogen) infection with possible roles ininflammation and inflammatory diseases (Partida-Sanchez et al., 2001;Normark et al., 2001), and synaptic plasticity (Reyes-Harde et al.,1999). Accordingly, the disease or condition associated with anADP-ribosyl transferase, ADP-ribosyl cyclase, ADP-ribosyl hydrolase, orNAD-dependent deacetylase enzyme also may include diseases or conditionsassociated with insulin release (e.g., diabetes), lymphocyte activation,bone homeostasis, and synaptic plasticity.

In these methods, the inhibitor or pro-drug compound may be chosen fromany of those previously described. Preferably, the inhibitor or pro-drugcompound is in a pharmaceutical composition and is one of the preferredcompounds previously described.

In the method of the present invention, an inhibitor or pro-drugcompound, as disclosed herein, is administered to a subject who has adisease or condition associated with an ADP-ribosyl transferase,ADP-ribosyl cyclase, ADP-ribosyl hydrolase, or NAD-dependent deacetylaseenzyme, in an amount effective to treat the disease or condition in thesubject. As used herein, the phrase “effective to treat the disease orcondition” means effective to ameliorate or mininmize the clinicalimpairment or symptoms resulting from the disease or conditionassociated with an ADP-ribosyl transferase, ADP-ribosyl cyclase,ADP-ribosyl hydrolase, or NAD-dependent deacetylase enzyme. For example,where the disease or condition is hypertension, the clinical impairmentor symptoms of the disease or condition may be ameliorated or minimizedby decreasing systolic and/or diastolic blood pressure, and therebyminimizing dizziness, flushed face, fatigue, headache, epistaxis,nervousness, and other symptoms associated with hypertension,particularly severe hypertension.

The amount of inhibitor or pro-drug compound effective to treat adisease or condition associated with an ADP-ribosyl transferase,ADP-ribosyl cyclase, ADP-ribosyl hydrolase, or NAD-dependent deacetylaseenzyme in a subject in need of treatment thereof will vary depending onthe particular factors of each case, including the type of disease orcondition associated with an ADP-ribosyl transferase, ADP-ribosylcyclase, ADP-ribosyl hydrolase, or NAD-dependent deacetylase enzyme, thesubject's weight, the severity of the subject's condition, and themethod of administration. Typically, the dosage for an adult human willrange from less than 1 mg to 1000 mg (preferably, 0.1 mg to 100 mg).Nevertheless, requisite amounts can be readily determined by the skilledartisan.

It is within the confines of the present invention that the inhibitor orpro-drug compounds disclosed herein may be administered to a subject whois already receiving an inhibitor of the ryanodine receptor or anantagonist that binds the ryanodine receptor. The inhibitor or pro-drugcompounds of the present invention, when contacted with an ADP-ribosyltransferase, cyclase, or hydrolase, or an NAD-dependent deacetylaseenzymes described herein, result in a decrease in cADPR concentration.It is expected that this decrease would prevent cADPR from competingagainst antagonists or inhibitors binding at the same site on theryanodine receptors.

In these methods, the inhibitor or pro-drug compound may be administeredto a human or animal subject by known procedures, including, withoutlimitation, oral administration, parenteral administration (e.g.,epifascial, intracapsular, intracutaneous, intradermal, intramuscular,intraorbital, intraperitoneal, intraspinal, intrasternal, intravascular,intravenous, parenchymatous, or subcutaneous administration),transdermal administration, and administration by osmotic pump.Preferably, the inhibitor or pro-drug compound of the present inventionis administered orally.

For oral administration, the inhibitor or pro-drug compound may beformulated in solid or liquid preparations, e.g., capsules, tablets,powders, granules, dispersions, solutions, and suspensions. Suchpreparations are well known in the art as are other oral dosage formsnot listed here. In a preferred embodiment, the inhibitor or pro-drugcompounds of the invention are tableted with conventional tablet bases,such as lactose, sucrose, mannitol, and corn starch, together with abinder, a disintegration agent, and a lubricant. These excipients arewell known in the art. The formulation may be presented with binders,such as crystalline cellulose, cellulose derivatives, acacia, cornstarch, or gelatins. Additionally, the formulation may be presented withdisintegrators, such as corn starch, potato starch, or sodiumcarboxymethylcellulose. The formulation also may be presented withdibasic calcium phosphate anhydrous or sodium starch glycolate. Finally,the formulation may be presented with lubricants, such as talc ormagnesium stearate. Other components, such as coloring agents andflavoring agents, also may be included. Liquid forms for use in theinvention include carriers, such as water and ethanol, with or withoutother agents, such as a pharmaceutically-acceptable surfactant orsuspending agent.

For parenteral administration (i.e., administration by injection througha route other than the alimentary canal), the inhibitor or pro-drugcompound may be combined with a sterile aqueous solution which ispreferably isotonic with the blood of the subject. Such a formulationmay be prepared by dissolving a solid active ingredient in watercontaining physiologically-compatible substances, such as sodiumchloride, glycine, and the like, and having a buffered pH compatiblewith physiological conditions, so as to produce an aqueous solution,then rendering said solution sterile. The formulations may be presentedin unit or multi-dose containers, such as sealed ampules or vials. Theformulation may be delivered by any mode of injection, including,without limitation, epifascial, intracapsular, intracutaneous,intradermal, intramuscular, intraorbital, intraperitoneal, intraspinal,intrasternal, intravascular, intravenous, parenchymatous, orsubcutaneous.

For transdermal administration, the inhibitor or pro-drug compound maybe combined with skin penetration enhancers, such as propylene glycol,polyethylene glycol, isopropanol, ethanol, oleic acid,N-methylpyrrolidone, and the like, which increase the permeability ofthe skin to the inhibitor or pro-drug compound, and permit the inhibitoror pro-drug compound to penetrate through the skin and into thebloodstream. The inhibitor or pro-drug compound/enhancer compositionalso may be further combined with a polymeric substance, such asethylcellulose, hydroxypropyl cellulose, ethylene/vinylacetate,polyvinyl pyrrolidone, and the like, to provide the composition in gelform, which may be dissolved in solvent, such as methylene chloride,evaporated to the desired viscosity, and then applied to backingmaterial to provide a patch. The inhibitor or pro-drug compound may beadministered transdermally, at or near the site on the subject where thedisease or condition is localized. Alternatively, the inhibitor orpro-drug compound may be administered transdermally at a site other thanthe affected area, in order to achieve systemic administration.

The inhibitor or pro-drug compound of the present invention also may bereleased or delivered from an osmotic mini-pump or other time-releasedevice. The release rate from an elementary osmotic mini-pump may bemodulated with a microporous, fast-response gel disposed in the releaseorifice. An osmotic mini-pump would be useful for controlling release,or targeting delivery, of the inhibitor or pro-drug compound.

In another aspect, the present invention provides methods of preparingthe inhibitor or pro-drug compounds described above. The methods mayinclude one or more of the methods disclosed herein, as well as othermethods that will be apparent to those of skill in the art. A method ofpreparing the inhibitors of the present invention may involve a reactionin the presence of silver, as an adaptation of several Hg²⁺ couplingsand chlorosugars to form nucleosides. In general, the methods willcomprise the following steps: (a) contacting a deoxyribose sugar (e.g.,β-3,5-bis-parachlorobenzoyl-1-pyridyl-2-deoxyribose), or a mixturecontaining a deoxyribose sugar and a base (e.g.,3,5-bis-parachlorobenzoyl-1-α-chloro-2-deoxyribose and nicotinamide),with a mixture containing both a silver compound (e.g., AgSbF₆) and thecompound to be reacted with the deoxyribose sugar (e.g., pyridine ornicotinanmide), thereby forming a reaction mixture; (b) redissolving thereaction mixture in MeOH; (c) adding NH₄Cl to the reaction mixture; (d)filtering the reaction mixture to remove precipitated residual silver;(e) treating the reaction mixture with NH₃ in MeOH; (f) adding water tothe reaction mixture; and (g) purifying the reaction mixture (e.g., withHPLC). See Example 1 for methods for preparation of particular inhibitorcompounds. Methods for the preparation of the pro-drug compounds of thepresent invention are provided in Example 2.

Preferred embodiments of the invention are described in the followingExamples. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered exemplary only, with the scope and spirit of the inventionbeing indicated by the claims which follow the examples.

EXAMPLE 1 Studies with Mechanism-Based Inhibitors of CD38

Example Summary

The soluble domain of human CD38 catalyzes the conversion of NAD⁺ tocyclic-ADP-ribose and to ADP-ribose via a common covalent intermediate(Sauve et al., 2000). Here we establish that mechanism-based inhibitorscan be produced by chemical stabilization of this intermediate. Thecompounds nicotinamide-2′-deoxyriboside (1),5-methyl-nicotinamide-2′-deoxyriboside (2) and pyridyl-2′-deoxyriboside(3) (FIG. 1) were synthesized and evaluated as inhibitors for humanCD38. The nicotinamide derivatives 1 and 2 were inhibitors of the enzymeas determined by competitive behavior in CD38 catalyzed conversion ofnicotiramide guanine dinucleotide (NGD⁺) to cyclic-GDP-ribose. The K_(i)values for competitive inhibition were 1.2 μM and 4.0 μM for 1 and 2respectively. Slow-onset characteristics of reaction progress curvesindicated a second higher affinity state of these two inhibitors.Inhibitor off-rates were slow with rate constants k_(off) of 1.5×10⁻⁵s⁻¹ for 1 and 2.5×10⁻⁵ s⁻¹ for 2. Apparent dissociation constantsK_(i(total)) for 1 and 2 were calculated to be 4.5 and 12.5 nMrespectively. The similar values for k_(off) are consistent with thehydrolysis of common enzymatic intermediates formed by the reaction of 1and 2 with the enzyme. Both form covalently attached deoxy-ribose groupsto the catalytic site nucleophile. Chemical evidence for thisintermediate is the ability of nicotinamide to rescue enzyme activityafter inactivation by either 1 or 2. A covalent intermediate is alsoindicated by the ability of CD38 to catalyze base exchange, as observedby conversion of 2 to 1 in the presence of nicotinamide. Thedeoxynucleosides 1 and 2 demonstrate that the chemical determinants formechanism-based inhibition of CD38 can be satisfied by nucleosides thatlack the 5′-phosphate, the adenylate group and the 2′-hydroxyl moiety.In addition, these compounds reveal the mechanism of CD38 catalysis toproceed by the formation of a covalent intermediate during normalcatalytic turnover with faster substrates. The covalent2′-deoxynucleoside inactivators of CD38 are powerful inhibitors byacting as good substrates for formation of the covalent intermediate butare poor leaving groups from the intermediate complex because hydrolyticassistance of the 2′-hydroxyl group is lacking. The removal of theadenylate nucleophile required for the cyclization reaction providesslow hydrolysis as the only exit from the covalent complex.

Reagents for chemical synthesis were obtained from commercial vendorsand were used as received. The synthesis of1-chloro-di-p-chloro-benzoyl-2-deoxyriboside (4), was synthesized asreported (Fox et al., 1961). This sugar could be stored with a P₂O₅sidearm for desiccation and stored at −78° C. [2′-3H]deoxyuridine wasobtained from ARC in 5 mCi quantity and used as received. Thymidinephosphorylase and alkaline phosphatase was obtained from Sigma. NMR datawas obtained on a Bruker DRX-300 instrument.

Synthesis of β-3,5-p-chlorobenzoyl-1-nicotinamide-2-deoxyriboside. 100mg (0.3 mmol) of 4 was added to a flame-dried flask containing 90 mg(0.8 mmol) nicotinamide. To a second flask, 20 mg (0.2 mmol)nicotinamide and 100 mg AgSbF₆ (0.3 mmol) was added, with 5 mLacetonitrile to dissolve the salt. The silver solution was cooled to 0°C. with ice and then added rapidly by syringe to the flask containingthe base and sugar. The solution was stirred chilled in an ice bath anda grayish precipitate formed. The reaction was stirred for 2 hourschilled and then warmed to room temperature and stirred an additional 2hours. The reaction mixture was evaporated, the residue redissolved inMeOH and filtered through Celite. The filtrate was evaporated. Thematerial was determined by NMR to contain the desired product in amixture of stereoisomers (9:1 β:α) in a yield of 85%. ¹HNMR, d₃-MeOD δ:(9.54 s, 1H), (9.25, s, 1H), (8.93, d, 1H), (8.2, m, 1H), (8.0-7.8, m,4H), (7.6-7.3, m, 4H), (6.79, t, 1H), (5.77, m, 1H), (5.01, m, 1H),(4.99-4.4, m, 3H), (3.44, m, 1H), (2.9, m, 1H).

Synthesis of β-nicotinamide-2′-deoxyriboside (1). The above material wassubjected to deprotection without further purification by treatment with5 mL 2 M NH₃ in MeOH at −20° C. This solution was reacted for 8 hours at−20° C. TLC was used to monitor the reaction. The MeOH and NH₃ wereevaporated at reduced pressure and the residue redissolved in 300 μL ofmethanol. 1 mL of water was then added. A gummy precipitate was removedby centrifugation and the aqueous phase was purified by HPLC to yieldpure α and β deprotected isomers of 1. These isomers were analyzed by ¹HNMR. Inhibitor solutions were measured at 266 nm for absorbance(concentration) and frozen upon isolation by HPLC and placed at −78° C.for later use. ¹H NMR, D₂O, δ: (9.5, s, 1H), (9.18, d, 1H), (8.84, d,1H), (8.16, t, 1H), (6.56, t, 1H), (4.47, m, 1H), (4.29, m, 1H),(4.54.0, m, 2H), (3.0, m, 1H), (2.82, m, 1H). MS: M⁺=239.

Synthesis ofβ-3,5-p-chlorobenzoyl-1-5-methyl-nicotinamide-2-deoxyriboside. 100 mg(0.3 mmol) of 4 was added to a flask along with 50 mg (0.4 mmol)5-methyl-nicotinamide. To this flask was added 2 mL CH₂Cl₂, and theflask kept on ice. To a second flask 50 mg (0.4 mmol)5-methyl-nicotinamide and 2 mL acetonitrile was added. The solution washeated to 50° C. to dissolve the 5-methyl-nicotinaide and subsequentlycooled to room temperature. 100 mg AgSbF₆ (0.3 mmol) was added and thesilver solution cooled to 0° C. with an ice bath. After several minuteson ice the contents of the silver solution were rapidly transferred bysyringe to the flask containing the base and sugar. The solution wasstirred while chilled by an ice bath and a grayish precipitate formed.The reaction was stirred for 2 hours chilled and then warmed to roomtemperature and stirred an additional 2 hours. The reaction mixture wasevaporated, the residue redissolved in MeOH and filtered through Celite.The filtrate was then evaporated. The material was determined by NMR tobe a mixture of stereoisomers in a ratio of 4.2:1 (β:α) in yield of 95%.¹HNMR, CD₃CN δ: (9.298, s, 1H), (9.017, s, 1H), (8.804, s, 1H), (8.275,d, 2H), (8.04, d, 2H), (7.77, d, 2H), (7.64, d, 2H), (6.78, t, 1H),(5.90, m, 1H), (5.178, m, 1H), (4.99-4.7, m, 2H), (3.44, m, 1H), (3.1,m, 1H). (2.66, s, 3H).

Synthesis of β-5-methyl-nicotinamide-2′-deoxyriboside (2). This materialwas subjected to deprotection without further purification by additionof 4 mL 2 M NH₃ in MeOH added at −20° C. and the reaction permitted togo for 8 hours at −20° C. The MeOH and NH₃ were then evaporated atreduced pressure and the residue redissolved in 1 mL of cold water.After trituration with water the suspension was spun to removeprecipitate and the aqueous phase purified by HPLC to yield the pure αand β deprotected isomers of 2. These isomers could be analyzed by ¹HNMR by rapid evaporation and redissolution in D₂O. Inhibitor solutionswere measured at 273 nm for absorbance (concentration) and frozen uponisolation by HPLC and placed at −78° C. ¹H NMR, D₂O, δ: (9.73, s, 1H),(9.43, s, 1H), (9.11, s, 1H), (6.86, t, 2H), (4.64, m, 1H), (4.11, m,1H), (4.04-3.78, m, 3H), (3.17, m, 1H), (2.96, s, 3H), (2.84, m, 1H).

Synthesis of β-3,5-p-chlorobenzoyl-1-pyridyl-2-deoxyriboside. 50 mg(0.15 mmol) of 4 was added to a flask. To a second flask was added 30 μLpyridine and 50 mg AgSbF₆ (0.15 mmol) and 5 mL acetonitrile/CH₂Cl₂ (1:4)was added to dissolve the salt. The silver solution was cooled to 0° C.with ice and then added to the flask containing the sugar. The solutionwas stirred chilled by ice bath and a precipitate was observed to form.The reaction was stirred for 2 hours chilled and then warmed to roomtemperature overnight. The reaction mixture was evaporated and theresidue redissolved in MeOH and filtered through Celite. The filtratewas then evaporated. The material was determined by NMR to be a mixtureof stereoisomers (14.3:1, β:α) in yield of 95%. ¹HNMR, CD₃CN δ: (9.19,d, 2H), (8.72, t, 1H), (8.27, t, 2H), (8.0-7.8, m, 4H), (7.7-7.5, m,4H), (6.88, t, 1H), (5.92, m, 1H), (6.78, t, 1H), (5.2, m, 1H), (4.9, m,1H), (3.4, m, 1H), (3.0, m, 1H).

Synthesis of β-pyridyl-deoxyriboside (3). The protected material abovewas subjected to deprotection without further purification by additionof 4 mL 2 M NH₃ in MeOH added at 0° C. and the reaction permitted to gofor 12 hours at 4° C. At the end of this time TLC indicated totalconsumption of starting material. The MeOH and NH₃ were then evaporatedat reduced pressure and the residue redissolved in 300 μL of methanolfollowed by addition of 1 mL of water. After trituration with water thesuspension was spun to remove precipitate and the aqueous phase purifiedby HPLC to yield the pure α and β deprotected isomers. 10.4:1 (β:α). ¹HNMR, D₂O, δ: (9.17, d, 2H), (8.72, t, 1H), (8.24, t, 2H), (6.66, t, 2H),(4.64, m, 1H), (4.44, dd, 1H), (4.04, dd, 1H), (3.92, dd, 1H), (2.99, m,1H), (2.74, m, 1H).

Determination of K_(i) and k_(on) by competitive method. To 1 mLsolutions of 50 mM potassium phosphate pH 7.2 and 100 μM NGD⁺containing, 50, 25, 12.5, and 6.25 and 0 μM inhibitor 1 was added 2 μLof 7 μM CD38. Reaction progress upon initiation by enzyme addition wasmonitored by measurement of 295 mm absorbance. The initial slopes wereused to determine the K_(i) value, and all points of the experiment werefit to the equation A(t)=vt+(b−v)(1−exp(−kt))/k+A₀ where k is theobserved rate constant, b is the initial rate, v is the final rate andA₀ is the initial absorbance was used to evaluate k_(on). A similarprocedure was used for inhibitor 2, with concentrations of componentsgiven in FIG. 3.

Off-Rate Measurement. 500 nM CD38 in 50 mM potassium phosphate pH 7.2was incubated with 15 μM inhibitor for 30 min at room temperature. 5 μLof the enzyme inhibitor solution was added to a cuvette containing 1 mLreaction of 50 mM potassium phosphate pH 7.5 containing 300 μM NGD⁺pre-chilled to 19° C. Production of NGD⁺ was determined by monitoring295 mm absorbance. The absorbance was fit to the equation A(t)=vt+(b−v)(1−exp(−kt))k+A₀ where A(t) is the absorbance, k is the rate constant ofrecovery, b is the initial rate, v is the final rate and A₀ is theinitial absorbance. A control lacking inhibitor but in all otherrespects identical was also run.

Radiochemical measurement of inhibitor binding. [2′-³H]Nicotinamidedeoxyriboside (1) was used to measure binding by the following method.Inhibitor at 9 μM with specific radioactivity of 866 cpm/nmol, wasincubated with 1.2 μM CD38 (monomer) in 1 mL 50 mM potassium phosphate(pH 7.5). The reactions were started by enzyme addition and quenched byfreezing with a dry ice/acetone bath at 30, 60 90, 120, 250, 500, and1000 s. Cooled (0° C.) gel filtration columns were used to separateprotein with cooled (0° C.) 10 mM potassium phosphate as eluant. Thefrozen fraction were quickly thawed, applied to these columns andfractions collected in 1 mL volumes over the course of several minutes.Scintillation fluid (9 mL) was then added to each 1 mL fraction andsamples counted. A sample lacking enzyme was performed as a blankcontrol as was a sample using the α-[2′-³H]nicotinamide deoxyriboside ofequal concentration and specific activity. The observed radioactivitywas fit using the equation A(t)=A₀(1−exp(−kt))+B. where A₀ is activityat reaction completion, k is the observed pseudo-first order rateconstant and B is the activity of the blank.

Activity recovery by addition of nicotinamide. 10 mL CD38 (1 μM) inK₂PO₄H was incubated with 2 (10 μM) for 30 minutes at room temperatureand subsequently placed on ice. Ten 1.5 mL solutions of NGD (400 μM)containing 0-100 mM nicotinamide were also prepared. To a two syringeApplied Photophysics spectrophotometer in fluorescence mode was addedinhibited enzyme to one syringe and NGD⁺ solution to the other.Fluorescence was used to monitor cGDPR formation and the totalfluorescence curves fit using the activity recovery equation.F(t)=vt+(b−v)(1−exp(−kt))/k+F₀ where k is the observed rate constant, bis the initial rate, v is the final rate and F₀ is the initialabsorbance. The value of k was plotted against the nicotinamideconcentration and the points fit to the Michaelis-Menten equation usingthe program Kaleidagraph.

Base-exchange reaction. 75 μM 2 was incubated with 1 μM CD38 enzyme andvarying concentrations of nicotinamide (0-40 mM) in 150 μL volumes.These reactions were run separately in autosampler tubes held at 19° C.in a temperature regulated autosampler and assayed by multiinjectionHPLC using 5 mM K₂PO₄H pH 5.0 and 2.5% MeOH as eluant. The quantity of 2reacted and the quantity of 1 formed versus time was determined byintegrations of the peaks for 1 and 2 with comparison to standards. Therate of conversion of 1 to 2 versus nicotinamide concentration wasplotted and the points fit to the Michaelis-Menten equation using theprogram Kaleidagraph.

Results

Synthesis of 2-deoxy-nicotinamide-ribosides. Several deoxy-nucleosidecompounds (1-3, FIG. 1) bearing 1′-β-pyridyl substitutions were preparedfrom the chloro-sugar 4 (Scheme 2). In the sugar-base coupling step astoichiometric quantity (versus sugar) of AgSbF₆ was found tosignificantly improve stereochemical yield of the β isomer and theoverall coupling yield. Standard deprotection protocol in coldmethanolic ammonia gave the desired derivatives in mixtures ofstereoisomers. Pure α and β stereoisomers were obtained bysemipreparative reverse phase HPLC.

Preparation of 2′-³H substituted versions of 1 and 2 were obtained byrepeating the syntheses above with [2-³H]2-deoxyribose. Thisradiolabeled sugar starting material was obtained by digestion ofcommercially available [2′-³H]2′-deoxyridine with the enzyme thymidinephosphorylase followed by treatment of the reaction mixture withalkaline phosphatase to form [2-³H]deoxyribose (Scheme 3). The specificradioactivity of the inhibitors was determined to be 866 cpm/nmol.

Determination of Competitive Inhibition by Initial Rates: The inhibitors1-3 were evaluated for inhibition of CD38 enzymatic activity using aspectrophotometric assay. CD38 catalyzes the conversion of NGD⁺ tocyclic-GDP ribose (cGDPR, Scheme 4) and product formation can bemonitored by 295 nm absorbance measurement. FIG. 2 shows the behavior of1 in assays using 100 μM NGD⁺ (50×K_(m), 35) and variable inhibitorconcentrations. The initial rates of these reactions (FIG. 2A)demonstrated competitive inhibition of CD38 cyclase activity by 1 with avalue for K_(i) of 1.2 μM±0.3. A similar reaction containing 40 μM NGD⁺at several concentrations of 2 was also performed (FIG. 3). Initialrates of reaction showed inhibition of CD38 cyclase activity by 2 with aK_(i) of 4.0±0.5 μM. Reaction mixtures containing 3, the α isomer of 1,or the α isomer of 2 did not inhibit CD38 conversion of NGD⁺ to cGDPReven at millimolar concentrations of these compounds (data not shown).

Slow Phase Inhibition. Reaction progress curves of CD38 activity inreaction mixtures containing the inhibitors 1 and 2 showed not onlyinitial rate inhibition but a second phase of slow-onset inhibitionindicated by slopes declining monotonically over time as seen in FIG. 2and FIG. 3. This slow phase was not due to substrate depletion and wasattributable to a kinetic process leading to progressive inhibition ofthe enzyme. The “slow-onset” absorbance curves could be fit using theequation A_(t)=v_(i)t+(v_(i)−v_(f))(1−exp(−kt))/k+A₀ where A_(t) isabsorbance, v_(i) is initial velocity, v_(f) is final velocity, t istime in s, k is the rate of the slow onset process and A₀ is absorbanceof the sample at initial time. These fits are shown by the solid linesin FIG. 2 and FIG. 3. The rate constant for the slow phase could beobtained from the average value of k determined from the separate fits.The value of k for 1 was determined to be 0.0042±0.001 s⁻¹ and the valueof k for 2 was determined to be 0.0083±0.002 s⁻¹. The parameter k isdefined as k_(on) in Scheme 5.

Recovery from Inhibition. To fully characterize the inhibition of 1 and2 against CD38, determinations of the inhibitor off-rates were needed.The inhibitor off-rate provides the final parameter in the equationk_(off)/k_(on)*K_(i)=K_(i(total)) which is valid for the kinetic schemeof slow-onset inhibition shown in Scheme 5. This rate was obtained by arecovery protocol in which inhibited CD38 enzyme (by either 1 or 2) wasadded to a 200 μM solution of NGD⁺. The solution 295 nm absorbance ismonitored spectrophotometrically to assay conversion of NGD⁺ to cGDPR asa consequence of regain of CD38 catlytic activity. Typical curvesobtained are shown in FIG. 4. The top curve shows product formation acontrol reaction using uninhibited CD38 and the bottom curve shows slowrecovery of activity of inhibited enzyme versus time. The absorbancecurves with the same equation used for slow-onset:A_(t)=v_(i)t+(v_(i)−v_(f))(1−exp(−kt))/k+A₀ where A_(t) is absorbance,v_(i) is initial velocity, v_(f) is final velocity, t is time in s, k isthe rate of the recovery rate constant and A₀ is the absorbance of thesample at initial time. The rate constant for the recovery phase wasobtained from the average value of k determined from the separate fits.The value of k_(off) for 1 was determined to be 1.5×10⁻⁵ s⁻¹ and for 2was determined to be 2.5×10⁻⁵ s⁻¹.

Calculation of Total Inhibition. Inhibition of CD38 by compounds 1 and 2could be fully described by the equationk_(off)/k_(on)*K_(i)=K_(i(total)) for the reactions described by Scheme5; where K_(i(total)) is the effective dissociation constant betweenfree CD38 and the fully inhibited complex. This equation is valid forslow onset behavior inhibitors and also mechanistic based inactivatorsof enzymes where a slow recovery of the enzyme from chemicalinactivation is present. Using the kinetic parameters in Table 2 thevalue for K_(i(total)) is 4.5 nM for inhibitor 1. Similarly, a value forK_(i(total)) of 12.5 nM was calculated for inhibitor 2. TABLE 2 Lineticand equilibrium parameters for inhibitors 1 and 2. Parameter 1 2 K_(i)1.2 μM  4.0 μM k_(on) 4.2 × 1O⁻³ s⁻¹ 8.3 × 1O⁻³ s⁻¹ k_(off) 1.5 × 1O⁻⁵s⁻¹ 2.5 × 1O⁻⁵ s⁻¹ K_(i(total)) 4.5 nM 12.5 nMAll values obtained at 19° C. The parameters are defined in accord withScheme 5. The value for K_(i(total)) was obtained from the relationK_(i(total)) = (k_(off)/k_(on))*K_(i). The values for each parameter inthe calculation is given in the table.

Nature of Inhibition in Slow Phase: Rescue by Base Addition. Our priorinvestigations of the nature of inhibition of CD38 by ara-F-NMN⁺ showedit to be governed by both competitive and slow-onset characteristics(Sauve et al., 2000). The slow onset behavior was shown to be aconsequence of covalent trapping of the catalytic nucleophile (Glu226)by the ara-F-sugar with nicotinamide leaving group departure. Byanalogy, the slow-onset inhibition of deoxy-nucleosides 1 and 2 isproposed to proceed via deoxyribose sugar transfer to the catalyticnucleophile. To test this hypothesis, additional chemical methods wereused to examine the scheme of inactivation shown in Scheme 6.

According to Scheme 6, recovery of enzymatic activity occurs via slowhydrolysis of the covalent intermediate to form deoxyribose and freeenzyme. If the covalent intermediate is the normal catalytic reactionpath, the enzyme catalytic activity should also be recovered frominhibition by reaction with a substrate nucleophile, such asnicotinamide (Scheme 7). The rate and equilibrium of the reaction willestablish the thermodynamic equilibrium of these species. For favorableequilibria, addition of product pyridine bases should permitbase-exchange reactions that will rapidly regenerate active enzyme.

Rescue of activity by substrate nucleophiles has been used as a test ofthe covalent mechanism in the ara-F-NMN⁺ inactivation of CD38, withrescue by nicotinamide (Sauve et al., 2000), and has been observed inthe covalent inactivation of adenosine nucleoside transferase (36) andother covalently inactivated glycosyl transferases (Withers, 2000).Here, the rescue experiment involved preincubation of CD38 with theinhibitor 2 followed by reaction of enzyme with a 300 μM solution ofNGD⁺ containing different concentrations of nicotinamide as aregenerating base. A stopped flow, two syringe fitted spectrophotometerwas used to perform the experiments. The fluorescence of cGDPR wasmeasured as a function of time to generate curves that contain anexponential and a linear phase prior to substrate exhaustion. Thesecurves were fit to the equationF_(t)=v_(f)t+(v_(i)−v_(f))(1−exp(−kt))/k+F₀ as previously defined. Therate constant k_(base) was plotted against the nicotinamideconcentration to obtain a saturation curve with an apparent K_(m) valuefor nicotinamide of 2.4 mM and a maximum rate of 0.023 s⁻¹ (FIG. 5). Thelimiting value of k_(base) as nicotinamide concentration is increasedindicates equilibrium binding of nicotinamide at the active site priorto chemical reaction. Therefore, the K_(m) represents an accuratemeasurement of K_(d) for nicotinamide for the covalent form of theenzyme. Similar saturation curves have been reported for the rescuebehavior of adenine on 2′-fluoro-adenosine inactivation of adenosinenucleoside transferase (Porter et al., 1995) and for nicotinamide rescueof CD38 from ara-F-NMN⁺ inactivation (Sauve et al., 2000).

Base exchange reaction. The nicotinamide rescue of CD38 enzymaticactivity inhibited by 2 completes a catalytic cycle that is proposed toeffect base exchange of 2 to form 1. The rescue reaction is thesecond-half of the normal reaction cycle of the base-exchange reactioncatalyzed by CD38, and inactivation of CD38 by 2 is the first half. Thishypothesis was tested by incubation of 75 μM 2, with varyingconcentrations of nicotinamide in the presence of 1 μM CD38 and thereactions monitored by HPLC. FIG. 6 shows that CD38 catalyzes theconversion of 2 to 1 under these conditions. The peak at 8 minuteselutes identically with authentic 1. The rates of these reactions couldbe monitored by multiple autosampler driven injections and the rates ofconversion plotted against nicotinamide concentration. The points werefit using the Michaelis-Menten equation (FIG. 7) to obtain a K_(m) of0.92 mM for nicotinamide and a k_(cat) of 0.007 s⁻¹. The observedk_(cat) value is within experimental error of the rate of inactivation(k_(out), 0.008 s⁻¹, Scheme 5) of CD38 by 2 measured at the sametemperature. This result establishes that the rate of intermediateformation is rate-limiting in the catalytic cycle of Scheme 7. Thegreater than two-fold lower K_(m) for steady state base exchange (0.92mM) versus the apparent K_(m) for nicotinamide rescue (2.4 mM) suggeststhat sub-maximal base binding to the nicotinamide binding pocket duringsteady state conditions is sufficient to maintain the maximum turnoverrate. This notion is supported by the rate constant for formation of thecovalent intermediate (k_(on), =0.008 s⁻¹) and the rate constant for thebase reaction step (k_(base)=0.023 s⁻¹) measured independently.

Titration of Enzyme with [2′-³H]Inhibitor. CD38 inactivation wasaccomplished using a radiolabeled inhibitor to assess inhibitorinteractions with the enzyme independent from inhibition of thecatalytic activity. This method allows the determination of thestoichiometry of covalent labeling, and detects cooperativity atmultiple sites. The labeling characteristics of CD38 show that it islabeled by 1 in a process governed by a single rate constant with avalue of k_(chem) 0.01 s⁻¹ at 25° C. (FIG. 8) within reasonableagreement with the rate constant for inactivation in kinetic assays ofinhibition (k_(on)=0.0042 s⁻¹ at 19° C.). The extent of labeling doesnot change after the first 20 minutes of incubation time, whichindicates that there is no non-specific labeling of the enzyme by theinhibitor. According to specific activity measurements and proteinconcentration, the labeling is 1:1 versus CD38 monomer concentration.This result is similar to what was observed with CD38 inhibition byara-F-NMN⁺ (Sauve et al., 2000).

Discussion

Deoxyriboside nicotinamide derivatives bind to the catalytic sites ofCD38 with higher binding affinity than the natural substrate, NAD⁺. 1has a binding constant of 1.2 μM and 2 has a binding constant of 4 μM.In comparison the K_(m) for dinucleotide and mononucleotide substratesis 150 μM for NMN⁺ (Sauve et al., 1998) 15 μM for NAD⁺ and 2.5 μM forNGD⁺. Based on these comparisons, it is apparent that truncation ofstructure does not necessarily weaken binding. Inhibition of CD38 by thedeoxynucleoside compounds 1 and 2 is not only competitive, but ischaracterized by a second kinetic phase of inhibition marked by enhancedaffinity of the inhibitor for the enzyme with rates of onset of 0.004s⁻¹ for 1 and 0.008 s⁻¹ for 2. Recovery of the enzyme from the secondphase of binding is quite slow, on the order of 2×10⁻⁵ s⁻¹ at 19° C.,and was similar within experimental error for both 1 and 2. By use ofthe equation for slow onset inhibitors,K_(i)k_(off)/k_(on)=K_(i(total)), the enhanced binding leads toinhibition values of 4.5 nM for inhibition of CD38 by 1 and 12.5 nM by2. These inhibition constants are lower than any known CD38 inhibitorsand confirm that highly abbreviated structures can rapidly and potentlyinhibit ADP-ribosyl cyclase enzymes. The magnitude of the rate constantsfor inactivation (k_(on)) show that tight inhibition can be achievedwithin minutes at physiological temperatures.

The nature of the slow-phase process leading to formation of the tightcomplex in Scheme 5 is proposed to be the covalent modification of theenzyme by inhibitor via attachment of the deoxyribose sugar to theenzyme catalytic nucleophile as shown in Scheme 6. A previous example ofcovalent modification of CD38 revealed that the inhibitor ara-F-NMN⁺forms a stable ara-F-ribose-5-phosphate ester with Glu226 (Sauve et al.,2000). MS studies confirmed the identity of this acid as the catalyticnucleophile. This residue is universally conserved across allADP-ribosyl cyclase sequences, and has been mutagenized with ablation ofcatalytic activity for CD38 (Munshi et al., 1999). The covalentmodification was shown to be reversible, and nicotinamide additions tothe trapped enzyme recover catalytic activity and reform ara-F-NMN⁺.Moreover, CD38 catalyzes base exchange using ara-F-NMN⁺ as a substrate.

Similar behaviors were observed for inhibited CD38 treated with thedeoxy-inhibitors 1 and 2. For instance, when fully inhibited CD38 enzyme(by either 1 or 2) was treated with millimolar concentrations ofnicotinamide, recovery of catalytic activity was observed, with a rateof recovery dependent on nicotinamide concentration. The recovery ratereached a saturable maximum versus nicotinamide concentration. Ratesaturation for recovery has also been observed in previous studies ofcovalent intermediates and the apparent K_(m) appears to reflect thebinding affinity of the rescue nucleophile within the active site. Thisvalue was found to be 17 mM for ara-F-NMN⁺ rescue by nicotinamide (Sauveet al., 2000), and the 2.4 mM value for rescue from the deoxyriboseintermediate indicates that nicotinamide binding is tighter in thecovalent complex formed by deoxy-ribose modification of CD38.

Scheme 7 shows inactivation and recovery as two mechanistic steps in acatalytic cycle that leads to inhibitor base exchange. Incubations of 2with nicotinamide confirmed that CD38 catalyzes base exchange to form 1and 5-methyl-nicotinamide. HPLC analysis was used to monitor thekinetics of base exchange because 1 is readily separated from 2 and baseexchange is fairly slow. The K_(m) of nicotinamide for base exchange is0.92 mM and the k_(cat) is 0.007 s⁻¹ at 19° C. The value of K_(m) forbase exchange (0.92 mM) is significantly lower than the value for theapparent K_(m) for base rescue (2.4 mM) of CD38 activity from inhibitionby 2. However, the rate of rescue saturates at 0.024 s⁻¹, suggesting thelower K_(m) for exchange takes its origin from chemistry of covalentmodification of the enzyme being rate limiting in the catalytic cycle,and because full site binding by base is unnecessary to maintain thesteady state rate in the second reaction of base exchange. Thus, therate of turnover of base exchange matches the rate of slow-phaseinhibitor inactivation by 2, which has the value 0.008 s⁻¹.

Radiochemical titrations of enzyme with [2′-³H]-1 confirm that labelingreaches maximum with a rate constant of 0.01 s⁻¹ and extendedincubations do not increase radiochemical labeling. The extent oflabeling is consistent with a ratio of inhibitor to subunit of 1:1.These measurements confirm that covalent modification is a specificprocess and is due to the covalent modification of the catalyticnucleophile responsible for catalysis with faster substrates.

The effectiveness of deoxy-nucleosides as trapping agents for CD38 is incontrast to chemical stability profiles. 2′-Deoxy derivatives areintrinsically more labile to uncatalyzed solvolysis reactions than thecorresponding ribose derivatives, and even more unstable than2′-fluorine substituted derivatives (Oppenhemer and Handlon, 1992).Trapping of covalent intermediates on nucleoside and glycosyltransferase enzymes has been successful in a number of cases by theintroduction of 2′-fluorine (Sauve et al., 2000; Porter et al., 1995;Withers, 2000), because of the electronic destabilization of thecationic charge that builds up at the anomeric carbon in transitionstates common to nucleoside and glycosyl transfer reactions. Theincrease in energy of these transition states retards breakdown oftrapped intermediates (Withers, 2000). In examination of the rate ofturnover of NMN⁺ versus ara-F-NMN⁺ on CD38 it was intriguing to note theratio of the rates of turnover of CD38 covalent intermediates was on theorder of 10⁷ at 37° C. (Sauve et al., 2000; Withers, 2000) which did notmatch the ratio of their uncatalyzed solvolysis rates, which wasmeasured to be 30:1 at the same temperature (Oppenhemer and Handlon,1992). This suggested that slow turnover of fluorine substitutedintermediates by CD38 took most of its origin from removal of the 2′-OHgroup and not from an electronic effect contributed by fluorine.

The results of this study strongly corroborate this viewpoint. In thiscase removal of the 2′-OH and replacement with a hydrogen atom inconjunction with removal of the 5′-phosphate group leads to efficientformation of the covalent intermediate but inefficient hydrolyticturnover of this species, leading to effective trapping and inhibitionof the enzyme. The role of the 2′-OH in catalysis is suggested by theproximity it normally has to the Glu226 leaving group which departs thesugar in intermediate breakdown. Both share the alpha face of the sugarand proton transfer to the Glu residue during catalytic turnover may bemediated in part by the 2′-OH. The proposed protonation state of thecatalytic Glu during catalysis is indicated in Scheme 6. Although thisrationale deserves additional investigation a strong Glu-2′OHinteraction has been proposed as a part of NAD⁺ glycohydrolase function(Oppenhemer and Handlon, 1992) and has recently been revealedcrystallographically in the enzyme BST-1 (Yamamoto-Katayama et al.,2002).

Trapping efficiency can also improve from the lack of the ADP group ininhibitors 1 and 2. The nucleoside structure lacks the adenylate of thenormal NAD⁺ substrate thus precluding the normal cyclization pathwayinherent to dinucleotide substrates. The absence of a reasonablenucleophile for an escape from the intermediate complex via theintramolecular route leaves hydrolysis as the only remaining escapepathway.

In conclusion, deoxynucleosides are effective mechanism based inhibitorsof CD38 through formation of a covalent intermediate that has beenidentified as part of the normal reaction coordinate of CD38 catalysis.A variety of techniques establish that this inactivation is specific toa single reactive moiety on the enzyme, which is catalytically competentto support the base exchange mechanism inherent to CD38 catalysis withfaster substrates. These compounds lack phosphate groups, have nanomolarbinding affinity for CD38 enzymes, inactivate within minutes at roomtemperature and have slow recovery rates, suggesting that thesederivatives may be effective probes of CD38 biochemical action in cellsand tissues.

EXAMPLE 2 Synthesis of Pro-Drugs of CD38 Inhibitors

Using the synthetic reaction sequence shown in Scheme 8 (see Sauve etal., 2002), three versions of compound IV (Scheme 8) were made. Theywere the compounds where (1) both X and Y are H; (2) X is methyl and Yis H; and (3) X is H and Y is methyl. With all three compounds, R isparachlorobenzine. The reaction sequence can be used for essentially anycompound where X and Y are each independently an alkyl, heteroatom orheterogroup, or H; and R is an alkyl or aryl group.

A utility of this flexible scheme is to furnish ester pro-drug forms ofthe free nucleoside that can be cleaved inside of cells by endogenousesterases (Scheme 9).

A utility of the ester modification is in the utilization of existingstrategies to synthesize the new pro-drug forms. The additionaladvantages gained include greater stability of the compound, better cellpermeability and activation only inside the target cell. This mayincrease the time of drug action by extending its lifetime inside thebody and may also increase potency by inhibiting degradation andincreasing target specific activity.

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantages attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

All references cited in this specification are hereby incorporated byreference. The discussion of the references herein is intended merely tosummarize the assertions made by the authors and no admission is madethat any reference constitutes prior art. Applicants reserve the rightto challenge the accuracy and pertinence of the cited references.

1-26. (canceled)
 27. A compound represented by the formula:

wherein A is a nitrogen-, oxygen-, or sulfur-linked aryl, alkyl, cyclic,or heterocyclic group; B is hydrogen, or a halogen, amino, or thiolgroup; C is hydrogen, or a halogen, amino, or thiol group; D is theester —OOCR where R is an alkyl or an aryl, a primary alcohol, ahydrogen, or an oxygen, nitrogen, carbon, or sulfur linked to phosphate,a phosphoryl group, a pyrophosphoryl group, or adenosine monophosphatethrough a phosphodiester or carbon-, nitrogen-, or sulfur-substitutedphosphodiester bridge, or to adenosine diphosphate through aphosphodiester or carbon-, nitrogen-, or sulfur-substitutedpyrophosphodiester bridge; and E is OH or the ester —OOCR where R is analkyl or an aryl, provided at least one of D or E is the ester —OOCRwhere R is an alkyl or an aryl.
 28. The compound of claim 27, whereinthe compound, when treated with an esterase, inhibits at least oneenzyme selected from the group consisting of an ADP-ribosyl transferase,an ADP-ribosyl cyclase, an ADP-ribosyl hydrolase, and an NAD-dependentdeacetylase enzyme.
 29. The compound of claim 28, wherein the enzyme isa CD38.
 30. The compound of claim 27, wherein A is further substitutedwith an electron contributing moiety
 31. The compound of claim 30,wherein the electron contributing moiety is selected from the groupconsisting of methyl, ethyl, O-methyl, amino, NMe₂, hydroxyl, CMe₃, aryland C3-C10 alkyl. 32-34. (canceled)
 35. The compound of claim 27,wherein A is capable of base exchange with nicotinamide in the presenceof a CD38.
 36. The compound of claim 27, wherein both D and E are theester —OOCR where R is an alkyl or an aryl.
 37. The compound of claim27, wherein A is an N-linked aryl or heterocyclic group.
 38. Thecompound of claim 27, wherein A is a substituted nicotinamide, pyrazolo,or imidazolo group.
 39. The compound of claim 27, which is amethyl-nicotinamide-2′-deoxyriboside ester.
 40. The compound of claim27, which is a 5-methyl-nicotinamide-2′-deoxyriboside ester or a4-methyl-nicotinamide-2′-deoxyriboside ester.
 41. The compound of claim27, which is an ester of β-1′-5-methyl-nicotinamide-2′-deoxyribose,β-D-1′-5-methyl-nicotinamide-2′-deoxyribofuranoside,β-1′-4-methyl-nicotinamide-2′-deoxyribose,β-D-1′-4-methyl-nicotinamide-2′-deoxyribofuranoside,β-1′-4,5-dimethyl-nicotinamide-2′-deoxyribose orβ-D-1′-4,5-dimethyl-nicotinamide-2′-deoxyribofuranoside.
 42. Thecompound of claim 27, which is an ester ofβ-1′-5-methyl-nicotinamide-2′-deoxyribose. 43-44. (canceled)
 45. Thecompound of claim 27, wherein both B and C are hydrogen, or either B orC is a halogen, amino, or thiol group and the other of B or C ishydrogen.
 46. The compound of claim 27, wherein D is a primary alcoholor hydrogen.
 47. A pharmaceutical composition comprising the compound ofclaim 28 and a pharmaceutically-acceptable carrier.
 48. A method forinhibiting an ADP-ribosyl transferase, ADP-ribosyl cyclase, ADP-ribosylhydrolase, or NAD-dependent deacetylase enzyme, comprising contactingthe enzyme with an esterase and an amount of the compound of claim 28effective to inhibit the enzyme.
 49. The method of claim 48, wherein theenzyme is a CD38.
 50. The method of claim 48, wherein the enzyme is in aviable mammalian cell.
 51. The method of claim 50, wherein the cell isin a mammal. 52-53. (canceled)